Phosphorylation of Smad2/3 at Specific Linker Threonine Indicates Slow-Cycling Intestinal Stem-Like Cells Before Reentry to Cell Cycle
Phosphorylation of Smad2/3 at Specific Linker Threonine Indicates Slow-Cycling Intestinal Stem-Like Cells Before Reentry to Cell Cycle
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DOI：
10.1007/s10620-014-3348-3
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发表时间：
2015-02-01
影响因子：
3.1
通讯作者：
Okazaki, Kazuichi
中科院分区：
文献类型：
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作者：
Kishimoto, Masanobu;Fukui, Toshiro;Okazaki, Kazuichi
静止（慢循环）和活跃（快循环）干细胞在小肠中被证明。我们在小鼠胃中发现了Smad 2/3（pSmad 2/3L-Thr）的显著表达，并认为这些细胞是上皮干细胞。在此，我们探讨了pSmad 2/3L-Thr是否可以作为小肠和结肠干细胞的生物标志物。我们检测了C57 BL/6小鼠的小肠和结肠以及葡聚糖硫酸钠（DSS）诱导的结肠炎。我们用Ki 67、细胞角蛋白8、嗜铬粒蛋白A、CDK 4、DCAMKL 1和Musashi-1对pSmad 2/3L-Thr进行双重免疫荧光染色。用pSmad 2/3L-Thr免疫荧光染色法检测Lgr 5-EGFP基因敲入小鼠小肠和结肠组织。为了检测pSmad 2/3L-Thr免疫染色阳性细胞的BrdU标记保留，我们收集了BrdU给药后的标本，观察了pSmad 2/3L-Thr与BrdU的双重免疫荧光染色。pSmad 2/3L-Thr与Ki 67的免疫组织化学共定位未观察到。pSmad 2/3L-Thr免疫染色强阳性细胞与细胞角蛋白8、CDK 4和Musashi-1共定位，与嗜铬粒蛋白A和DCAMKL 1免疫染色阳性细胞定位不同。光镜下pSmad 2/3L-Thr免疫染色强阳性细胞形态未分化。在Lgr 5-EGFP基因敲入小鼠中，部分但不是全部pSmad 2/3L-Thr免疫染色强阳性细胞显示与Lgr 5共定位。pSmad 2/3L-Thr免疫染色强阳性细胞在给药后5、10和15天显示与BrdU共定位。在DSS诱导的结肠炎中，pSmad 2/3L-Thr和Ki 67免疫染色阳性细胞在再生期增加，在损伤期减少，提示pSmad 2/3L-Thr免疫染色强阳性细胞是即将进入细胞周期的上皮干细胞样细胞。
Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells.Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells.We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU.In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase.In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.